System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations

ABSTRACT

Devices, compositions, and methods for handling, separating, packaging, and utilization of spermatozoa (1) that can be derived from previously frozen sperm samples collected from a male mammal. Specifically, techniques to uniformity stain (2) spermatozoal DNA even when derived from previously frozen sperm and separation techniques to separate and isolate spermatozoa even when derived from previously frozen sperm samples into X-chromosome bearing and Y-chromosome bearing populations having high purity.

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/253,787, filed Nov. 29, 2000 and U.S. ProvisionalPatent Application No. 60/253,785, filed Nov. 29, 2000, each herebyincorporated by reference herein.

I. TECHNICAL FEILD

[0002] The invention involves the substantially uniform binding offluorochrome(s) to the DNA within mammalian spermatozoa (or sperm cells)allowing such labeled spermatozoa to be separated into high purityX-chromosome bearing and Y-chromosome bearing populations. Specifically,methods for the substantially uniform binding of fluorochrome(s) to theDNA of mammalian spermatozoa contained within previously frozen and thenthawed semen. In addition, the invention further involves devices,methods, and compositions for the use of high purity separatedX-chromosome bearing and Y-chromosome bearing populations of spermatozoafrom previously frozen-thawed semen in processes involving, but notlimited to, artificial insemination, surgical insemination, and in-vitrofertilization and embryo culturing techniques.

II. BACKGROUND

[0003] Sperm can be collected from a great variety of mammals and thenseparated into X-chromosome bearing and Y-chromosome bearing populationsbased upon the difference in DNA content. In some conventional methodsof spermatozoa separation, the DNA content of the spermatozoa to beseparated can be stained with a fluorochrome(s) that upon excitationemit(s) a measurable amount of fluorescence. Because X-chromosomebearing spermatozoa contain a greater amount of DNA than Y-chromosomebearing spermatozoa, each X-chromosome bearing spermatozoa has thecapacity to bind a relatively greater amount of fluorochrome than thecorresponding Y-chromosome bearing spermatozoa. Comparison of therelative magnitude of emitted fluorescence upon excitation of thefluorochrome(s) allows the isolation of X-chromosome bearing spermatozoafrom Y-chromosome bearing spermatozoa as described by U.S. Pat. No.5,135,759, hereby incorporated by reference.

[0004] Even though X-chromosome bearing spermatozoa and Y-chromosomebearing spermatozoa have been differentiated by and separated based uponthe difference in emitted fluorescence for many years, and even thoughthere is large commercial market for isolated populations ofX-chromosome bearing spermatozoa and Y-chromosome bearing spermatozoa,there remain significant problems yet to be resolved.

[0005] A significant problem with conventional methods of separatingX-chromosome bearing spermatozoa from Y-chromosome bearing spermatozoacan be that each resulting population contains a significant number ofincorrectly separated spermatozoa that belong in the other population.This problem in differentiating between spermatozoa can, in part, beattributed to the lack of uniformity in the amount of fluorochrome boundto the spermatozoal DNA. As such, a range in the amount of fluorochromebound by X-chromosome bearing spermatozoa is generated and a range inthe amount of fluorochrome bound by Y-chromosome bearing spermatozoa isgenerated. When these ranges in the amount of fluorochrome overlap oryield some values that are similar, it can be difficult or impossible toclassify those individual spermatozoa to one population or the otherwith any degree of certainty and cross contamination of the populationscan occur.

[0006] This particular problem can be exacerbated with regard tospermatozoa obtained from frozen and subsequently thawed mammaliansemen. The mean purity for separated Y-chromosome bearing spermatozoapopulation derived from previously frozen-thawed semen can be 85% orless, and the mean purity for separated X-chromosome bearing spermatozoapopulation derived from previously frozen-thawed semen can be 82% orless.

[0007] Another significant problem associated with staining ofspermatozoal DNA can be the detrimental effects on fertilization ratesand subsequent embryonic development of fertilized oocyte(s) (oocyte,ootid, or ovum, or a plurality of same, as may be appropriate within aspecific application). One aspect of this problem may be that the amountof stain bound to the DNA may effect the viability of the spermatozoaresulting in lower fertilization rates. Another aspect of this problemcan be that the amount of time that elapses during the staining of theDNA may effect the viability of the sperm resulting in lowerfertilization rates. Another aspect of this problem may be that theamount of time that elapses during staining of the DNA may lowersubsequent cleavage rates of oocytes fertilized with such stainedspermatozoa. A 20% decline in cleavage rates have been observed foroocytes when staining time requires 190 minutes as compared to whenstaining time requires 60 minutes. Another aspect of this problem may bethat the percent of oocytes fertilized with stained spermatozoa thatproceed to blastulation may be lower as described in the journal articleentitled “In vitro Fertilization with Flow-Cytometrically-Sorted BovineSperm”, Theriogenology 52: 1393-1405 (1999), hereby incorporated byreference herein.

[0008] Another significant problem may be that cryopreserved sperm maydemostrate increased capacitation, and the length of time suchspermatozoa are viable may be shortened. As such, if previously frozenspermatozoa are to be separated into X-chromosome bearing andY-chromosome bearing populations that are to be subsequently used inapplications such as in-vitro fertilization, in-vivo artificialinsemination, or the like, then routine staining procedures may have tobe abbreviated to maintain suitable number of viable sperm cells.

[0009] As relating to the problems of staining spermatozoa uniformly,even when spermatozoa are obtained from previously frozen-thawed semen;maintaining sperm viability; separating stained spermatozoa intoX-chromosome bearing and Y-chromosome bearing populations, even when thespermatozoa being separated are obtained from previously frozen semen;generating populations of X-chromosome bearing and Y-chromosome bearingspermatozoa having high purity; and successfully using separatedspermatozoa for artificial insemination, surgical insemination, andin-vitro fertilization techniques it can be understood there aresignificant problems with conventional technology which are addressed bythe instant invention.

III. DISCLOSURE OF THE INVENTION

[0010] A broad object of embodiments of the invention can be to provideDNA staining technology that allows substantially uniform amounts offluorochrome to be bound to the DNA of all individual spermatozoabearing an X-chromosome and substantially uniform amounts offluorochrome to be bound to all individual spermatozoa bearing aY-chromosome within an amount of semen.

[0011] One aspect of this broad object of the invention can be to narrowthe range in magnitude of emitted fluorescence for each of theX-chromosome bearing population and the Y-chromosome bearing populationof spermatozoa upon passing through a fluorochrome excitation source.

[0012] Another aspect of this broad object of the invention can be toincrease the difference between the mean values of magnitude of emittedfluorescence for each of the X-chromosome bearing population and theY-chromosome bearing population of spermatozoa upon passing through afluorochrome excitation source.

[0013] Another aspect of this broad object of the invention can be todecrease the number of spermatozoa incorrectly assigned to each of theX-chromosome bearing population and the Y-chromosome bearing populationof spermatozoa.

[0014] Another aspect of this broad object of the invention can be togenerate separate X-chromosome bearing and Y-chromosome bearingpopulations having greater than 85% purity or greater than 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%purity.

[0015] Another broad object of embodiment of the invention can be toallow assessment of a wide range of genetics. Rather than being limitedto the genetics of individuals from species of mammals having proximityto a spermatozoa separating or sorting facility, genetics representing awide variety of individuals from numerous species can be transported asfrozen semen to distant spermatozoa separation facilities for subsequentseparation into X-chromosome bearing and into Y-chromosome bearingpopulations. These species of mammals may include, but are not limitedto primates, such as chimpanzees, gorillas, humans, or the like; marinemammals, such as whales, porpoises, or the like; bovids; ovids; swine;canids; felids; or equids, as but a few examples. It may also includegenetics that are considered rare because the species of mammal may beendangered or few in number; or considered rare because the individualhas desirable morphological, physiological, or intellectual attributes.

[0016] Another broad object of embodiments of the invention can provideseparation technology for differentiating between X-chromosome bearingand Y-chromosome bearing spermatozoa obtained from frozen-thawed semen.

[0017] Another object of embodiments of the invention can be to provideDNA staining technology to more uniformly stain the DNA of spermatozoacontained in frozen-thawed semen to improve the apparent resolutionbetween X-chromosome bearing and Y-chromosome bearing spermatozoa.

[0018] Another object of embodiments of the invention can be to providehigh purity artificial insemination samples prepared from separatedspermatozoa from frozen-thawed semen.

[0019] Another object of embodiments of the invention can be to providehigh purity low dose artificial insemination samples prepared fromseparated spermatozoa from frozen-thawed semen.

[0020] Another object of embodiments of the invention can be to providehigh purity insemination samples for surgical insemination proceduresprepared from separated spermatozoa from frozen-thawed semen.

[0021] Another object of an embodiment of the invention can be toprovide high purity insemination samples for in-vitro fertilizationprocedures prepared from separated spermatozoa from frozen-thawed semen.

[0022] Another object of an embodiment of the invention can be toprovide high purity insemination samples for in-vitro fertilizationprocedures prepared from separated spermatozoa from frozen-thawed humansemen.

[0023] Another object of an embodiment of the invention can be toprovide technology for staining and separation of spermatozoa fromfrozen-thawed sperm into X-chromosome bearing populations andY-chromosome bearing populations for in-vitro fertilization of oocyte(s)that is not detrimental to cleavage rates or embryonic development.

[0024] Naturally further objects of the invention are disclosedthroughout other areas of specification.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 shows a particular embodiment of the invention for stainingthe DNA of spermatozoa contained in frozen-thawed semen.

[0026]FIG. 2 shows a particular embodiment of the invention forseparating spermatozoa from frozen-thawed semen into X-chromosomebearing and Y-chromosome bearing spermatozoa.

[0027]FIG. 3 shows a further view of a particular embodiment of theinvention for separating spermatozoa from frozen-thawed semen intoX-chromosome bearing and Y-chromosome bearing spermatozoa.

V. MODE(S) FOR CARRYING OUT THE INVENTION

[0028] To routinely separate spermatozoa (live, fixed, viable,non-viable, or nuclei) into high purity X-chromosome bearing samples andinto Y-chromosome bearing samples, the method used to sort theX-chromosome bearing and Y-chromosome bearing spermatozoa must providesufficient resolution of the X-chromosome bearing spermatozoa from theY-chromosome bearing spermatozoa so that separation or sorting step(s)can be achieved without substantial cross contamination.

[0029] Resolution or differentiation of spermatozoa can be based uponascertaining the difference in the fluorescent emission from the amountof fluorochrome bound to the DNA within the X-chromosome bearingspermatozoa upon excitation and the fluorescent emission from the amountof fluorochrome bound to the DNA within the Y-chromosome bearingspermatozoa upon excitation. Separation of X-chromosome bearingspermatozoa and Y-chromosome bearing spermatozoa based upon thismeasurable difference may then be achieved by a number of methods suchas flow cytometry, liquid chromatography, gel electrophoresis, and othertechnologies that similarly compare the relative magnitude offluorescence to differentiate between X-chromosome bearing spermatozoaand the Y-chromosome bearing spermatozoa.

[0030] Spermatozoa separation systems can have problems differentiatingbetween the fluorescent emission generated by the fluorochrome bound tothe DNA of X-spermatozoa, and the fluorescent emission generated by thefluorochrome bound to the DNA of Y-spermatozoa upon excitation when theamount of the fluorochrome bound to the DNA of individual spermatozoa isnot consistent within the Y-chromosome bearing or X-chromosome bearingpopulations. These difficulties in differentiating between the amount offluorescent emissions generated by the bound fluorochrome(s) becomeexacerbated when spermatozoa are obtained from frozen-thawed sperm whichare stained by conventional techniques.

[0031] The failure to stain the spermatozoal DNA consistently cangenerate a broader range of fluorescing species for both X-chromosomebearing and Y-chromosome bearing populations of spermatozoa. Thisbroader range of fluorescing species for the two populations results inan increased range of apparent DNA molecular weights and a decreasedability to resolve X-chromosome bearing from Y-chromosome bearingspermatozoa. The decrease in resolution makes separation of theX-chromosome bearing spermatozoa from the Y-chromosome bearingspermatozoa more difficult and results in cross contamination betweenpopulations and a lower purity of separated spermatozoa samples areobtained.

[0032] Particular embodiments of the invention provide technology tostain the DNA of live viable spermatozoa or the spermatozoal DNA offrozen-thawed semen specimens to allow increased resolution ofX-chromosome bearing from the Y-chromosome bearing spermatozoa resultingin high purity X-chromosome bearing and high purity Y-chromosome bearingpopulations of sperm cells. As such, it is understood that the term highpurity can mean greater resolution of the X-chromosome bearing from theY-chromosome bearing spermatozoa compared to conventional stainingtechnology for a given application. High purity can also mean less crosscontamination between separated spermatozoa populations compared toconventional separation technologies.

[0033] For example, in particular flow cytometry embodiments of theinvention, high purity for stained frozen-thawed live spermatozoa canmean sorted populations of X-chromosome bearing spermatozoa andY-chromosome bearing spermatozoa having a purity greater than about 85%.However, if live viable sperm or sperm nuclei are being sorted highpurity may mean X-chromosome bearing and Y-chromosome bearingpopulations having a purity greater than about 90%. As can beunderstood, the definition of high purity is contextual involving acomparison of the results obtained from each embodiment of the inventioncompared to the results obtained when utilizing convention technologiesfor a particular application. In the context of spermatozoa having DNAthat stains poorly, such as previously frozen-thawed spermatozoal DNA,high purity can mean populations of isolated spermatozoa bearing greaterthan 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%, of either an X-chromosome or a Y-chromosome.

[0034] Embodiments of the invention can include spermatozoa collectedfrom numerous species of male mammals, and the invention should beunderstood not to be limited to the species of male mammals described bythe specific examples within this application. Rather the specificexamples within this application are intended to be illustrative of thevaried and numerous species of male mammals from which semen can becollected and utilized in certain embodiments of the invention.Embodiments of the invention, for example, may include the spermatozoaof animals having commercial value for meat or dairy production such asswine, ovids, bovids, equids, buffalo, or the like (naturally themammals used for meat or dairy production may vary from culture toculture). It may also include the spermatozoa of various domesticatedmammalian species encompassed by canids and felids. It may also includespermatozoa from individuals of various mammalian species that haveuncommon attribute(s), such as morphological characteristics includingweight, size, or conformation, or other desired characteristics such asspeed, agility, intellect, or the like. It may also include spermatozoaof primates, including but not limited to chimpanzees, gorillas, orhumans and the spermatozoa from marine mammals such as whales anddolphins. It may also include frozen-thawed spermatozoa from all thevarious mammals above-described and further, including but not limitedto, the spermatozoa of deceased donors, from rare or exotic mammals,zoological specimens, or endangered species.

[0035] Now referring primarily to FIG. 1, particular embodiments of theinvention can comprise semen containing spermatozoa (1) collected from amale mammal, including but not limited to, those above-described. Thespermatozoa can be incubated in a concentration of Hoechst 33342 stain(2) of greater than about 40 μM at a temperature between about 30°Centigrade and about 40° Centigrade for a duration of time between 50minutes to 200 minutes to stain spermatozoal DNA with sufficientuniformity to allow X-chromosome bearing spermatozoa to bedifferentiated from Y-chromosome bearing spermatozoa based upon themagnitude of fluorescence at a rate greater than about 85%.

[0036] The concentration of Hoechst 33342 stain between 40 μM and 2500μM, the temperature between 30° Centigrade and about 40° Centigrade, andthe duration of time between 50 minutes and 200 minutes can be selectedto adjust the purity of the separated X-chromosome bearing andY-chromosome bearing populations, or can be selected to promote cleavagerates and embryonic development, as further discussed below.

[0037] For example, when staining spermatozoal DNA from certain bovinespecies, the concentration of Hoechst 33342 can be increased to betweenabout 200 μM and about 2500 μM, incubated for a period of time betweenabout 60 minutes to about 190 minutes at a temperature of about 37°Centigrade. Specifically with respect to certain frozen-thawed bovinespermatozoa, the Hoechst 33342 stain (2) can be adjusted to establish aconcentration of 2240 μM and then incubated for about 60 minutes atabout 39° Centigrade.

[0038] With respect to the cleavage rates of oocytes inseminated withmammalian sperm cells treated according to the invention, the increasein stain concentration up to at least 2240 μM does not appear to have adepressive effect on either cleavage or embryonic development. Higherstain concentrations may actually be beneficial with respect to certainembodiments of the invention because the length of incubation time maybe decreased improving percent cleavage or blastocyst formation. Fromapplication to application the concentration of Hoechst 33342, thelength of incubation time, or both can be adjusted to obtain the maximalcleavage rate and blastocyst formation, if desired.

[0039] Now referring primarily to FIGS. 2 and 3, flow cytometricembodiments of the invention can include a cell source (3) which acts toestablish or supply stained spermatozoa (fresh, frozen-thawed, spermnuclei, or the like) to be analyzed by flow cytometry. The cells aredeposited within a nozzle (4) in a manner such that the stained spermcells are surrounded by a sheath fluid (5). The sheath fluid (5) isusually supplied by a sheath fluid source (6) so that as the cell source(3) supplies sperm cells, the sheath fluid (5) is concurrently fedthrough the nozzle (4). In this manner the sheath fluid (5) forms asheath fluid environment for the sperm cells. Since the various fluidsare provided to the flow cytometer at some pressure, they flow out ofthe nozzle (4) and exit at the nozzle orifice (7). By providing a typeof oscillator (8) which may be very precisely controlled through anoscillator control (9), pressure waves may be established within thenozzle (4) and transmitted to the fluids exiting the nozzle (4) at thenozzle orifice (7). Since the oscillator (9) acts upon the sheath fluid(5), the stream (10) exiting the nozzle orifice (7) eventually andregularly forms drops (11). Because the sperm cells are at leastpartially surrounded by a sheath fluid environment, the drops (11) cancontain within them individually isolated sperm cells.

[0040] Since the drops (11) generally contain individual isolated spermcells, the flow cytometer can distinguish and separate droplets basedupon the magnitude of fluorescence emitted from the fluorochrome boundto the spermatozoal DNA. This is accomplished through a cell sensingsystem (12). The cell sensing system involves at least some type ofsensor (13) which responds to the magnitude of fluorescence emitted byeach sperm cell contained within each drop (11). The sperm cell sensingsystem (13) may cause an action depending upon the relative presence orrelative absence of fluorescence emitted by the bound fluorochrome uponexcitation by some stimulant such as the laser exciter (14). While eachspermatozoon can be stained by the fluorochrome, such as Hoechst 33342,as described above, the differing amount of DNA comprising theX-chromosome and the Y-chromosome causes different amounts of stain tobe bound. Thus, by sensing the degree of fluorescence emitted by thefluorochrome upon excitation it is possible to discriminate betweenX-bearing spermatozoa and Y-bearing spermatozoa by their differingemission levels.

[0041] In order to achieve separation and isolation of the appropriatesperm cells, the signals received by sensor (14) are fed to some type ofsorter discrimination system (15) which very rapidly makes adifferentiation decision and can differentially charge each drop (11)based upon whether it has decided that the desired sperm cell does ordoes not exist within that drop (11). In this manner the separation ordiscrimination system (15) acts to permit the electrostatic deflectionplates (16) to deflect drops (11) based on whether or not they containthe appropriate sperm cell. As a result, the flow cytometer acts to sortcells by causing them to land in one or more collectors or containmentelements (17). Thus by sensing some property of the sperm cells (such asmagnitude of fluorescense), the flow cytometer can discriminate betweensperm cells based on that particular characteristic and place them inthe appropriate collector or containment element (17). In particularembodiments of the invention using flow cytometry to sort spermatozoa,the X-bearing sperm cell containing droplets are charged positively andthus deflect in one direction, and the Y-bearing sperm cell containingdroplets are charged negatively and thus deflect the other way, and thewasted stream (containing unsortable sperm cells) remain uncharged andthus can be collected in an undeflected stream into a suction tube, orthe like.

[0042] Now referring primarily to FIG. 3, the nozzle (4) emits a stream(10) which because of the oscillator (8) (not shown in FIG. 3) formsdrops (11). Since the sperm cell source (3) (not shown in FIG. 3) maysupply sperm cells (1) which may be stained according to theabove-described invention, the light emission from the boundfluorochrome excited by laser exciter (13) can be differentiallydetermined by sensor (14) so that the existence or nonexistence of acharge on each drop (11) as it separates from stream (10) can becontrolled by the flow cytometer. This control results in positivelycharged, negatively charged, or uncharged drops (8) based upon the spermcell contained within each drop (11). As shown by FIG. 3, certain dropsare shown as deflected drops (18). These deflected drops (18) are thosecontaining spermatozoon differentiated by bearing either an X-chromosomeor a Y-chromosome. Separated spermatozoa are then isolated in anappropriate collection element or containment element (17) for lateruse.

[0043] Embodiments of the invention can comprise droplets (11) eachcontaining a sperm cell (15) bearing either an X-chromosome or aY-chromosome. Droplets containing X-chromosome bearing sperm cells canbe isolated into containment element(s) (17) at a rate of at least 1000per second or at a rate greater than about 1000 per second, such as 2000per second, 3000 per second, 4000 per second, 5000 per second, orhigher. Similarly Y-chromosome bearing sperm cells can be isolated at arate of at least 1000 per second or at a rate greater than about 1000per second, such as 2000 per second, 3000 per second, 4000 per second,5000 per second, or higher. In some embodiments of the invention,droplets containing X-chromosome bearing sperm cells and dropletscontaining Y-chromosome bearing sperm cells are simultaneously separatedand isolated into containment elements each at a rate of at least 1000per second, or greater than 1000 per second, such as 2000 per second,3000 per second, 4000 per second, 5000 per second, or at even higherrates.

[0044] Embodiments of the invention can also include artificialinsemination samples prepared from sperm cells collected from malemammals (which can be frozen and thawed with respect to some embodimentsof the invention) that are then stained and separated according toembodiments of the invention above-described. The artificialinsemination samples can then be utilization in artificial inseminationprotocols. For example, a bovine artificial insemination sample preparedfrom separated spermatozoa according to the invention can comprise fewerthan 10×10⁶ viable spermatozoa contained within a straw. Low doseartificial insemination samples for bovine artificial insemination cancontain as few as 1-3×10⁶ viable spermatozoa, or even as few as 150,000spermatozoa as described in U.S. patent application Ser. No. 09/001,394,or PCT Patent Application US98/27909, each hereby incorporated byreference. Artificial insemination samples, having a regular number ofseparated sperm cells or a low dose of separated sperm cells can be usedin animal breeding programs, such as those described in U.S. PatentApplications No. 60/224,050 and No. 60/21,093, each hereby incorporatedby reference. Artificial insemination samples containing previouslyfrozen and thawed spermatozoa stained and separated according to theinvention can also be utilized in conjunction with synchronized breedingprograms using superovulated animals as described in U.S. patentapplication Ser. No. 09/001,454, hereby incorporated by referenceherein. Naturally, for frozen sperm cells that are of limitedavailability because the male mammal is deceased, or the male mammal isa rare or exotic animal, an artificial insemination sample preparedaccording to the invention may contain even fewer spermatozoa.

[0045] The number of viable separated spermatozoa that are stained,separated, and isolated into X-chromosome bearing or Y-chromosomebearing populations according to the invention that are used in anartificial insemination sample can vary based upon the species of mammalto be artificially inseminated. For example, equine artificialinsemination samples prepared from separated spermatozoa may require ahigher number of viable separated spermatozoa relative to the bovineapplication, as described in PCT Patent Application US99/17165, herebyincorporated by reference. An embodiment of an equine inseminationsample may, as but one example, contain between about forty million toabout one-hundred million spermatozoa.

[0046] In certain embodiments of the invention, the insemination samplecontaining separated spermatozoa collected from a male mammal orobtained from frozen-thawed sperm may be packaged for use with surgicalinsemination procedures

[0047] Sperm cells stained, separated, or isolated according to theinvention can also be used to fertilize oocyte(s) in-vitro (IVF). Anattractive feature of IVF can be that fewer separated sperm are needthan for artificial insemination. It may be desirable to use the fewestsperm possible, especially if the male mammal is deceased, rare, orexotic or if the spermatozoa are stained or separated in accordance withvarious embodiments of the invention. Also, commercial availability ofsperm cells separated into X-chromosome bearing and Y-chromosome bearingpopulations, especially when the male mammal is located a distance fromthe female mammal, or is exotic, rare, or has desirable attributes, willlikely result in greatly expanded use of IVF in breeding programs.Certain embodiments of the invention can include devices andmethodologies for the use of separated spermatozoa, including but notlimited to frozen-thawed sperm cells, with respect to the in-vitrofertilization of oocytes, the in-vitro oocyte maturation, or thein-vitro culture of zygotes, such as those described in the journalarticle by Lu, K. H., Cran D. G., and Seidel, G. E., In-vitroFertilization With Flow Cytometrically-Sorted Bovine Sperm,Theriogenology, 52, 1393-1405 (1999), hereby incorporated by reference.

[0048] Certain embodiments of the invention involving the production orgeneration of mammalian embryos can comprise collection of semen (1)from a male mammal or obtaining semen or spermatozoa (1) that are orhave been previously frozen. According to embodiments of the inventiondescribed above, the semen is combined with Hoechst 33342 (2) stain toestablish a concentration of between 40 μM and 2500 μM. The sperm cellsare incubated with the Hoechst 33342 stain at a temperature betweenabout 30° Centigrade and about 40° Centigrade for a duration of betweenabout 50 minutes to about 200 minutes. The stained sperm cells may beseparated and isolated into X-chromosome bearing and Y-chromosomebearing populations according to embodiments of the invention describedabove or by other sperm cell separation techniques that alsodifferentiate X-chromosome bearing spermatozoa from Y-chromosome bearingspermatozoa based upon the magnitude of fluorescence. The isolated spermcells may then be used to fertilize oocytes from a female mammal of thesame species, and in some cases from female mammals of differentspecies, in-vitro.

[0049] As an example of an application of embodiments of the inventioninvolving frozen bull sperm in IVF applications, sperm samples from twobulls were stained either at a concentration of 224 μM or 2,240 μM ofHoechst 33342 and the stained spermatozoa were then bulk sorted on aflow cytometer at 1000 sperm/sec into 2% egg yolk citrate. Spermatozoawere inseminated at 1×10⁶/mL and embryos were cultured in the mSOFsystem described by Tervit H. R. et al., Successful Culture In-Vitro ofSheep and Cattle Ova, J. Reprod. Fertil., 30:493-497 (1992), herebyincorporated by reference. Three replicates were carried out for bull 1and one replicate for bull 2 (Table 1). TABLE 1 Effect of stainconcentration on cleavage and developmental rates of oocytes inseminatedwith separated stained spermatozoa from frozen-thawed sperm. HoechstStaining 33342 time % No. conc. required No. blastocysts/ BullEjaculates (μM) (min) oocytes % cleave oocyte 1 3 224 190 368 44^(a) 171 3 2240 60 373 60^(b) 23 2 1 224 190 86 23^(a)   0^(a) 2 1 2240 60 8142^(b)  16^(b)

[0050] As can be understood, It can take much longer to stainfrozen-thawed sperm so that they can be resolved during separation atthe lower stain concentration than at 10× stain concentration. Thedifferences observed in cleavage rates between the two stainconcentrations most likely can be attributed to the extended incubationtime at the lower stain level. It appears that a 10-fold increase instain concentration does not have depressive effect on either cleavageof embryonic development.

[0051] As can be easily understood from the foregoing, the basicconcepts of the present invention may be embodied in a variety of ways.It involves the staining of spermatozoa, whether fresh spermatozoa orfrozen-thawed spermatozoa, separation and isolation techniques which maybe used with such stained spermatozoa, as well as devices to accomplishthe staining, separation, and isolation of such stained spermatozoa intoX-chromosome bearing and Y-chromosome bearing populations. In thispatent application, the staining and separating techniques used withspermatozoa are disclosed as part of the results shown to be achieved bythe various devices described and as steps which are inherent toutilization. They are simply the natural result of utilizing the devicesas intended and described. In addition, while some devices aredisclosed, it should be understood that these not only accomplishcertain methods but also can be varied in a number of ways. Importantly,as to all of the foregoing, all of these facets should be understood tobe encompassed by this disclosure.

[0052] The discussion included in this international Patent CooperationTreaty patent application is intended to serve as a basic description.The reader should be aware that the specific discussion may notexplicitly describe all embodiments possible; many alternatives areimplicit. It also may not fully explain the generic nature of theinvention and may not explicitly show how each feature or element canactually be representative of a broader function or of a great varietyof alternative or equivalent elements. Again, these are implicitlyincluded in this disclosure. Where the invention is described infunctionally-oriented terminology, each aspect of the function isaccomplished by a device, subroutine, or program. Apparatus claims maynot only be included for the devices described, but also method orprocess claims may be included to address the functions the inventionand each element performs. Neither the description nor the terminologyis intended to limit the scope of the claims which now be included.

[0053] Further, each of the various elements of the invention and claimsmay also be achieved in a variety of manners. This disclosure should beunderstood to encompass each such variation, be it a variation of anembodiment of any apparatus embodiment, a method or process embodiment,or even merely a variation of any element of these. Particularly, itshould be understood that as the disclosure relates to elements of theinvention, the words for each element may be expressed by equivalentapparatus terms or method terms—even if only the function or result isthe same. Such equivalent, broader, or even more generic terms should beconsidered to be encompassed in the description of each element oraction. Such terms can be substituted where desired to make explicit theimplicitly broad coverage to which this invention is entitled. As butone example, it should be understood that all actions may be expressedas a means for taking that action or as an element which causes thataction. Similarly, each physical element disclosed should be understoodto encompass a disclosure of the action which that physical elementfacilitates. Regarding this last aspect, as but one example, thedisclosure of a “sorter” should be understood to encompass disclosure ofthe act of “sorting”—whether explicitly discussed or not—and,conversely, were there only disclosure of the act of “sorting”, such adisclosure should be understood to encompass disclosure of a “sorter”and even a “means for sorting”. Such changes and alternative terms areto be understood to be explicitly included in the description.Additionally, the various combinations and permutations of all elementsor applications can be created and presented. All can be done tooptimize the design or performance in a specific application.

[0054] Any acts of law, statutes, regulations, or rules mentioned inthis application for patent: or patents, publications, or otherreferences mentioned in this application for patent are herebyincorporated by reference. Specifically, U.S. Provisional PatentApplication No. 60/253,787, filed Nov. 29, 2000 and U.S. ProvisionalPatent Application No. 60/253,785, filed Nov. 29,2000, are herebyincorporated by reference including any figures or attachments, and eachof references in the following table of references are herebyincorporated by reference. US Patent Documents DOCUMENT NO. 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M., AFRC Animal Breeding ResearchOrganisation, West Mains Road, Edinburg EH9 3JQ, “Efficiency of FoodUtilization in Traditional and Sex-Controlled Systems of BeefProduction”, pp 401-440. Taylor, S. C. S., A. J. Moore, R. B. Thiessen,and C. M. Bailey. “Efficiency of food utilization in traditional andsex-controlled systems of beef-production.” Animal Production 40: 401.1985 Tervit, H. R., et al., “Successful Culture In Vitro of Sheep andCattle Ova”, Agricultural Research Council, Unit of ReproductionPhysiology and Biochemistry, University of Cambridge, 1972, p. 493-497.Unruh, J. A. “Effects of endogenous and exogenous growth-promotingcompounds on carcass composition, meat quality and meatnutritional-valu˜.” J. Anim. Sci. 62: 1441. 1986 U.S. application Ser.No. 09/454,488, entitled “Improved Flow Cytometer Nozzle and FlowCytometer Sample Handling Methods”, filed Dec. 3, 1999. U.S. applicationSer. No. 60/238,294, entitled “Hysteroscopic Insemination of Mares”filed Oct. 5, 2000. 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Vazquez, J., et al., “SuccessfulLow-Dose Insemination by a Fiberoptic Endoscope Technique in the Sow“,Proceedings Annual Conference of the International Embryo TransferSociety, Netherlands, Theriogenology, Vol. 53, January, 2000, p. 201.Vazquez, J., et al., “Hypoosmotic Swelling Test as Predictor of theMembrane Integrity in Boar Spermatozo”, Boar Semen Preservation IV, IVthInternational Conference on Boar Semen Preservation, Maryland, pp. 263.Vidament, M., Dupere, A. M., Julienne, P., Evain, A., Noue, P. andPalmer, E. 1997. Equine frozen semen freezeability and fertility fieldresults. Theriogenology. 48: 907. Vincent, B. C., S. D. M. Jones, L. E.Jeremiah, M. A. Price, and J. A. Newman. “Carcass characteristics andmeat quality of once-calved heifers.” Canadian J. Anim. Sci. 71: 311.1991 Voss, J. L. and Pickett, B. W. 1976. Reproductive management of thebroodmare. C.S.U. Exp. Sta. Anim. Reprod. Lab. Gen. Series. Bull. 1-12Voss, J. L., Pickett, B. W., Burwash, L. D. and Daniels, W. H. 1974.Effect of human chorionic gonadotropin on duration of estrous cycle andfertility of normally cycling, nonlactating mares. J.A.V.M.A. 165:704-706. Voss, J. L., Squires, E. L., Pickett, B. W., Shideler, R. K.and Eikenberry, D. J. 1982. Effect of number and frequency ofinseminations on fertility in mares. J. Reprod. Fertil. Suppl. 32:53-57. Waggoner, A. W., M. E. Dikeman, I. R. Brethour, and K. E. Kemp.“Performance, carcass, cartilage calcium, sensory and collagen traits oflongissimus muscles of open versus 30-month-old heifers that producedone calf.” I. Anim. Sci. 68: 2380. 1990 Welch G. R., et al., 1994.Fluidic and optical modifications to a FACS IV for flow sorting of X-and Y- chromosome bearing sperm based on DNA. Cytometry 17 (suppl. 7):74. Welch, G., et al., “Flow Cytometric Sperm Sorting and PCR to ConfirmSeparation of X- and Y-Chromosome Bearing Bovine Sperm□, AnimalBiotechnology, 6(2), 131-139, 1995, pp 131-139. Wheeler, T. L., L. v.Cundiff and R. M. Koch. “Effect of marbling degree on beef palatabilityin Bos-Taurus and Bos-Indicus cattle.” J. Anim. Sci. 72: 3145. 1994Wickersham, E. W. and L. H. Schultz. “Infilience of age at firstbreeding on growth, reproduction, and production ofwell-fed holsteinheifers.” J. Dairy Sci. 46: 544. 1963 Wilson, C. G., Downie, C. R.,Hughes, J. P. and Roser, J. F. 1990. Effects of repeated hCG injectionson reproductive efficiency in mares. Eq. Vet. Sci. 4: 301-308. Wilson,M. S. 1993. Non-surgical intrauterine artificial insemination in bitchesusing frozen semen. J. Reprod. Fert Suppl. 47: 307-311. Woods, J. andGinther, O. J. 1983. Recent studies related to the collection ofmultiple embryos in mares. Theriogenology. 19: 101-108. Woods, J.,Bergfelt, D. R. and Ginther, O. J. 1990. Effects of time of inseminationrelative to ovulation on pregnancy rate and embryonic-loss rate inmares. Eq. Vet. J. 22(6): 410-415. XP-002103478, File Biosis, one page.

[0055] In addition, as to each term used it should be understood thatunless its utilization in this application is inconsistent with suchinterpretation, common dictionary definitions should be understood asincorporated for each term and all definitions, alternative terms, andsynonyms such as contained in the Random House Webster's UnabridgedDictionary, second edition are hereby incorporated by reference.However, as to each of the above, to the extent that such information orstatements incorporated by reference might be considered inconsistentwith the patenting of this/these invention(s) such statements areexpressly not to be considered as made by the applicant(s).

[0056] In addition, unless the context requires otherwise, it should beunderstood that the term “comprise” or variations such as “comprises” or“comprising”, are intended to imply the inclusion of a stated element orstep or group of elements or steps but not the exclusion of any otherelement or step or group of elements or steps. Such terms should beinterpreted in their most expansive form so as to afford the applicantthe broadest coverage legally permissible in countries such as Australiaand the like.

[0057] Thus, the applicant(s) should be understood to have support toclaim at least: i) each of the staining, separation, isolation,insemination, or fertilization procedures as herein disclosed anddescribed, ii) the related methods disclosed and described, iii)similar, equivalent, and even implicit variations of each of thesedevices and methods, iv) those alternative designs which accomplish eachof the functions shown as are disclosed and described, v) thosealternative designs and methods which accomplish each of the functionsshown as are implicit to accomplish that which is disclosed anddescribed, vi) each feature, component, and step shown as separate andindependent inventions, vii) the applications enhanced by the varioussystems or components disclosed, viii) the resulting products producedby such systems or components, ix) methods and apparatuses substantiallyas described hereinbefore and with reference to any of the accompanyingexamples, and x) the various combinations and permutations of each ofthe elements disclosed.

[0058] The claims set forth in this specification are herebyincorporated by reference as part of this description of the invention,and the applicant expressly reserves the right to use all of or aportion of such incorporated content of such claims as additionaldescription to support any of or all of the claims or any element orcomponent thereof, and the applicant further expressly reserves theright to move any portion of or all of the incorporated content of suchclaims or any element or component thereof from the description into theclaims or vice-versa as necessary to define the subject matter for whichprotection is sought by this application or by any subsequentcontinuation, division, or continuation-in-part application thereof, orto obtain any benefit of, reduction in fees pursuant to, or to complywith the patent laws, rules, or regulations of any country or treaty,and such content incorporated by reference shall survive during theentire pendency of this application including any subsequentcontinuation, division, or continuation-in-part application thereof orany reissue or extension thereon.

I claim:
 1. A method of staining sperm cells collected from mammals,comprising the steps of: a. collecting semen from a male mammal; b.incubating sperm cells contained within said semen in a concentration ofHoechst 33342 stain of greater than 40 micro-molar; c. establishing thetemperature at which said sperm cells in said concentration of Hoechst33342 stain are incubated between about 30 degrees centigrade and about40 degrees centigrade; d. adjusting a duration of time said sperm cellsare incubated with said concentration of Hoechst 33342 stain betweenabout 50 minutes and about 200 minutes; and e. staining DNA within saidsperm cells with sufficient uniformity to allow X-chromosome bearingsperm cells to be differentiated from Y-chromosome bearing sperm cellsbased upon the magnitude of fluorescence at a rate of greater than about85%.
 2. A method of staining sperm cells collected from mammals asdescribed in claim 1, wherein said male mammal is selected from thegroup of mammals consisting of primates, humans, swine, ovids, bovids,equids, canids, felids, and dolphins.
 3. A method of staining spermcells collected from mammals as described in claim 2, wherein said malemammal comprises said bovid and said concentration of Hoechst 33342stain is between about 200 micro-molar and about 2500 micro-molar.
 4. Amethod of staining sperm cells collected from mammals as described inclaim 2, wherein said male mammal comprises said bovid and saidconcentration of Hoechst 33342 stain is 224 micro-molar.
 5. A method ofstaining sperm cells collected from mammals as described in claim 2,wherein said male mammal comprises said bovid and said concentration ofHoechst 3342 stain is 2240 micro-molar.
 6. A method of staining spermcells collected from mammals as described in claim 4, wherein said stepof adjusting a duration of time said sperm cells are incubated with saidconcentration of Hoechst 33342 stain between about 50 minutes and about200 minutes comprises adjusting said duration of time said sperm cellsare incubated with said concentration of Hoechst 33342 stain to about190 minutes.
 7. A method of staining sperm cells collected from mammalsas described in claim 5, wherein said step of adjusting a duration oftime said sperm cells are incubated with said concentration of Hoechst33342 stain between about 50 minutes and about 200 minutes comprisesadjusting said duration of time said sperm cells are incubated with saidconcentration of Hoechst 33342 stain to about 60 minutes.
 8. A method ofstaining sperm cells collected from mammals as described in claim 1,wherein said step of staining DNA within said sperm cells withsufficient uniformity to allow X-chromosome bearing sperm cells to bedifferentiated from Y-chromosome bearing sperm cells based upon themagnitude of fluorescence at a rate of greater than about 85% comprisesdifferentiating said Y-chromosome bearing sperm cells from saidX-chromosome bearing sperm cells based upon the magnitude offluorescence at a rate selected from the group consisting of 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
 9. Amethod of staining sperm cells collected from mammals as described inclaim 8, wherein said X-chromosome bearing sperm cells differentiatedfrom said Y-chromosome bearing sperm cells comprise viable sperm cells.10. A method of staining sperm cells collected from mammals as describedin claims 1, 2, 6, 7, 8, or 9, further comprising the step of freezingsaid semen.
 11. A method of staining sperm cells collected from mammalsas described in claim 10, further comprising the step of thawing saidsemen.
 12. A method of staining sperm cells collected from mammals asdescribed in claim 11, wherein said step of staining DNA within saidsperm cells with sufficient uniformity to allow X-chromosome bearingsperm cells to be differentiated from Y-chromosome bearing sperm cellsbased upon the magnitude of fluorescence at a rate of greater than about85% comprises differentiating said magnitude of fluorescence with a flowcytometer.
 13. A method of staining DNA within frozen-thawed spermcells, comprising the steps of: a. collecting semen containing spermcells from a male mammal; b. freezing said semen containing said spermcells; c. thawing said semen containing said sperm cells; d. combiningfrozen-thawed semen with Hoechst 33342 stain; e. establishing aconcentration of said Hoechst 33342 stain between about 200 micro-molarand about 2500 micro-molar; f. adjusting the temperature of saidfrozen-thawed semen in said concentration of Hoechst 33342 stain tobetween about 30 degrees Centigrade and about 40 degrees Centigrade; andg. adjusting the duration of time said frozen-thawed semen in saidconcentration of Hoechst 33342 stain incubates to between about 50minutes and 200 minutes, whereby DNA within sperm cells contained insaid frozen-thawed are stained with sufficient uniformity todifferentiate between X-chromosome bearing sperm cells and Y-chromosomebearing sperm cells on the basis of magnitude of fluorescence.
 14. Amethod of staining sperm cells collected from mammals as described inclaim 13, wherein said male mammal is selected from the group of mammalsconsisting of primates, humans, swine, ovids, bovids, equids, canids,felids, and dolphins.
 15. A method of staining sperm cells collectedfrom mammals as described in claim 14, wherein said male mammalcomprises said bovid and said concentration of Hoechst 33342 stain isbetween about 200 micro-molar and about 2500 micro-molar.
 16. A methodof staining sperm cells collected from mammals as described in claim 14,wherein said male mammal comprises said bovid and said concentration ofHoechst 33342 stain is 224 micro-molar.
 17. A method of staining spermcells collected from mammals as described in claim 14, wherein said malemammal comprises said bovid and said concentration of Hoechst 3342 stainis 2240 micro-molar.
 18. A method of staining sperm cells collected frommammals as described in claim 14, wherein said step of adjusting aduration of time said sperm cells are incubated with said concentrationof Hoechst 33342 stain between about 50 minutes and about 200 minutescomprises adjusting said duration of time said sperm cells are incubatedwith said concentration of Hoechst 33342 stain to about 190 minutes. 19.A method of staining sperm cells collected from mammals as described inclaim 17, wherein said step of adjusting a duration of time said spermcells are incubated with said concentration of Hoechst 33342 stainbetween about 50 minutes and about 200 minutes comprises adjusting saidduration of time said sperm cells are incubated with said concentrationof Hoechst 33342 stain to about 60 minutes.
 20. A method of stainingsperm cells collected from mammals as described in claims 13, 15, 16,17, 18, 19, or 20, wherein said step of staining DNA within said spermcells with sufficient uniformity to allow X-chromosome bearing spermcells to be differentiated from Y-chromosome bearing sperm cells basedupon the magnitude of fluorescence at a rate of greater than about 85%comprises differentiating said Y-chromosome bearing sperm cells fromsaid X-chromosome bearing sperm cells based upon the magnitude offluorescence at a rate selected from the group consisting of 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
 21. Amethod of staining sperm cells collected from mammals as described inclaim 20, wherein said X-chromosome bearing sperm cells differentiatedfrom said Y-chromosome bearing sperm cells comprise viable sperm cells.22. A method of staining sperm cells collected from mammals as describedin claim 20, wherein said step of staining DNA within said sperm cellswith sufficient uniformity to allow X-chromosome bearing sperm cells tobe differentiated from Y-chromosome bearing sperm cells based upon themagnitude of fluorescence at a rate of greater than about 85% comprisesdifferentiating said magnitude of fluorescence with a flow cytometer.23. A method of generating mammalian embryos, comprising the steps of:a. collecting semen from a male mammal; b. combining said semen fromsaid male mammal with an amount of Hoechst 33342 stain; c. establishinga concentration of Hoechst 33342 stain combined with said semen to aconcentration between about 40 micro-molar and about 2500 micro-molar;d. adjusting the temperature at which said sperm cells are incubatedwith said Hoechst 33342 stain between about 30 degrees centigrade andabout 40 degrees centigrade; e. adjusting the duration of time saidsperm cells are incubated in said concentration of Hoechst 33342 betweenabout 60 minutes and about 200 minutes; f. staining DNA within spermcells contained in said semen with said Hoechst 33342 stain; and g.fertilizing oocytes with stained sperm cells, whereby increasing theconcentration of Hoechst 33342 stain and decreasing the duration of timesaid sperm cells are incubated in said concentration of Hoechst 33342stain increases the percentage of said mammalian embryos produced.
 24. Amethod of staining sperm cells collected from mammals as described inclaim 23, wherein said male mammal is selected from the group of mammalsconsisting of primates, humans, swine, ovids, bovids, equids, canids,felids, and dolphins.
 25. A method of staining sperm cells collectedfrom mammals as described in claim 24, wherein said male mammalcomprises said bovid and said concentration of Hoechst 33342 stain isbetween about 200 micro-molar and about 2500 micro-molar.
 26. A methodof staining sperm cells collected from mammals as described in claim 24,wherein said male mammal comprises said bovid and said concentration ofHoechst 33342 stain is 224 micro-molar.
 27. A method of staining spermcells collected from mammals as described in claim 24, wherein said malemammal comprises said bovid and said concentration of Hoechst 3342 stainis 2240 micro-molar.
 28. A method of staining sperm cells collected frommammals as described in claim 26, wherein said step of adjusting aduration of time said sperm cells are incubated with said concentrationof Hoechst 33342 stain between about 50 minutes and about 200 minutescomprises adjusting said duration of time said sperm cells are incubatedwith said concentration of Hoechst 33342 stain to about 190 minutes. 29.A method of staining sperm cells collected from mammals as described inclaim 27, wherein said step of adjusting a duration of time said spermcells are incubated with said concentration of Hoechst 33342 stainbetween about 50 minutes and about 200 minutes comprises adjusting saidduration of time said sperm cells are incubated with said concentrationof Hoechst 33342 stain to about 60 minutes.
 30. A method of stainingsperm cells collected from mammals as described in claims 23, 25, 26,27, 28, 29, or 30, wherein said step of staining DNA within said spermcells with sufficient uniformity to allow X-chromosome bearing spermcells to be differentiated from Y-chromosome bearing sperm cells basedupon the magnitude of fluorescence at a rate of greater than about 85%comprises differentiating said Y-chromosome bearing sperm cells fromsaid X-chromosome bearing sperm cells based upon the magnitude offluorescence at a rate selected from the group consisting of 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
 31. Amethod of staining sperm cells collected from mammals as described inclaim 30, wherein said X-chromosome bearing sperm cells differentiatedfrom said Y-chromosome bearing sperm cells comprise viable sperm cells.32. A method of staining sperm cells collected from mammals as describedin claim 30, wherein said step of staining DNA within said sperm cellswith sufficient uniformity to allow X-chromosome bearing sperm cells tobe differentiated from Y-chromosome bearing sperm cells based upon themagnitude of fluorescence at a rate of greater than about 85% comprisesdifferentiating said magnitude of fluorescence with a flow cytometer.33. A method of staining sperm cells collected from mammals as describedin claim 32, further comprising the step of isolating differentiatedX-chromosome bearing sperm cells and Y-chromosome bearing sperm cellsinto separate collection elements.
 34. A method of staining sperm cellscollected from mammals as described in claim 33, wherein said step ofisolating differentiated X-chromosome bearing sperm cells andY-chromosome bearing sperm cells into separate collection elementscomprises isolating Y-chromosome bearing sperm cells into a separatecollection element at a rate of about 1000 per second.
 35. A method ofstaining sperm cells collected from mammals as described in claim 33,wherein said step of isolating differentiated X-chromosome bearing spermcells and Y-chromosome bearing sperm cells into separate collectionelements comprises isolating X-chromosome bearing sperm cells into aseparate collection element at a rate of about 1000 per second.
 36. Amethod of generating mammalian embryos as described in claims 23,further comprising the step of freezing said semen.
 37. A method ofgenerating mammalian embryos as described in claims 30, furthercomprising the step of freezing said semen.
 38. A method of generatingmammalian embryos as described in claim 36, further comprising the stepof thawing said semen.
 39. A method of generating mammalian embryos asdescribed in claim 37, further comprising the step of thawing saidsemen.
 40. A flow cytometer system for isolating desired sperm cells,comprising: a. sperm cells obtained by thawing previously frozen semen,wherein said sperm cells are incubated with a concentration of Hoechst33342 stain between about 200 micro-molar and about 2500 micro-molaruntil DNA within said sperm cells are stained with sufficient uniformityto allow X-chromosome bearing sperm cells to be differentiated fromY-chromosome bearing sperm cells based upon the magnitude offluorescence at a rate of greater than about 85%; b. a sperm cell sourcethat supplies said sperm cells to a flow cytometer; c. a sheath fluidsource that creates a sheath fluid environment within said flowcytometer in which said sperm cells are entrained; d. a nozzle throughwhich said sperm cells pass while entrained in said sheath fluidenvironment; e. an oscillator that acts upon said sheath fluid as itpasses through said nozzle; f. a sperm cell sensing system responsive tosaid sperm cells; g. a separation discrimination system that acts toseparate said sperms cells having a desired characteristic; and h. acontainment element into which said sperm cells having said desiredcharacteristic are collected.
 41. A flow cytometer system for isolatingdesired sperm cells as described in claim 40, wherein said sperm cellsobtained by thawing previously frozen semen are obtained from malemammals selected from the group consisting of primates, humans, swine,ovids, bovids, equids, canids, felids, and dolphins.
 42. A flowcytometer system for isolating desired sperm cells as described in claim41, wherein said male mammal comprises said bovid, and wherein saidconcentration of Hoechst 33342 stain is between about 200 micro-molarand about 2500 micro-molar.
 43. A flow cytometer system for isolatingdesired sperm cells as described in claim 41, wherein said male mammalcomprises said bovid, and wherein said concentration of Hoechst 33342stain is about 224 micro-molar.
 44. A flow cytometer system forisolating desired sperm cells as described in claim 41, wherein saidmale mammal comprises said bovid, and wherein said concentration ofHoechst 3342 stain is 2240 micro-molar.
 45. A flow cytometer system forisolating desired sperm cells as described in claim 42, furthercomprising a duration of time said sperm cells are incubated with saidconcentration of Hoechst 33342 stain between about 50 minutes and about200 minutes.
 46. A flow cytometer system for isolating desired spermcells as described in claim 43, wherein said duration of time about 190minutes.
 47. A flow cytometer system for isolating desired sperm cellsas described in claim 44, wherein said duration of time about 60minutes.
 48. A flow cytometer system for isolating desired sperm cellsas described in claims 40, 42, 43, 45, or 48, wherein said X-chromosomebearing sperm cells to be differentiated from Y-chromosome bearing spermcells based upon the magnitude of fluorescence at a rate of greater thanabout 85% is a rate selected from the group consisting of 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
 49. A flowcytometer system for isolating desired sperm cells as described in claim48, further comprising a collection element into which differentiatedX-chromosome bearing sperm cells are isolated.
 50. A flow cytometersystem for isolating desired sperm cells as described in claim 48,further comprising a collection element into which differentiatedY-chromosome bearing sperm are isolated.
 51. A flow cytometer system forisolating desired sperm cells as described in claim 49, furthercomprising a rate at which X-chromosome bearing sperm cells are isolatedgreater than about 1000 per second.
 52. A flow cytometer system forisolating desired sperm cells as described in claim 50, furthercomprising a rate at which Y-chromosome bearing sperm cells are isolatedgreater than about 1000 per second.
 53. A method of generating mammalianembryos as described in claim 40, further comprising the step offreezing said semen.
 54. A method of generating mammalian embryos asdescribed in claims 51, further comprising the step of freezing saidsemen.
 55. A method of generating mammalian embryos as described inclaim 53, further comprising the step of thawing said semen.
 56. Amethod of generating mammalian embryos as described in claim 54, furthercomprising the step of thawing said semen.
 57. A method of producing amammal having a predetermined sex comprising the steps of: a. collectingsemen from a male mammal; b. freezing said semen; c. thawing said semen;b. determining the sex characteristic of a plurality of sperm cellscontained within said frozen-thawed semen; c. separating said spermcells according to the determination of their sex characteristic; d.isolating sperm cells separated according to the determination of theirsex in a collection element; d. establishing an artificial inseminationsample from said sperm cells isolated in said collection element; e.inserting said artificial insemination sample into a female mammal ofthe same species from which said semen was collected; f. fertilizing atleast one egg within said female mammal; and g. producing an offspringmammal of the desired sex.
 58. A method of producing a mammal having apredetermined sex as described in claim 57, wherein said male mammal isselected from the group of mammals consisting of primates, humans,swine, ovids, bovids, equids, canids, felids, and dolphins.
 59. A methodof producing a mammal having a predetermined sex as described in claim58, further comprising the step of staining DNA within said sperm cellswith a concentration of Hoechst 33342 greater than 40 micro-molar.
 60. Amethod of producing a mammal having a predetermined sex as described inclaim 59, wherein said step of staining DNA within said sperm cells witha concentration of Hoechst 33342 greater than 40 micro-molar comprisesstaining of sufficient uniformity to allow X-chromosome bearing spermcells to be differentiated from Y-chromosome bearing sperm cells basedupon the magnitude of fluorescence at a rate of greater than about 85%.61. A method of producing a mammal having a predetermined sex asdescribed in claim 59, wherein said male mammal comprises said bovid,and wherein said concentration of Hoechst 33342 stain is between about200 micro-molar and about 2500 micro-molar.
 62. A method of producing amammal having a predetermined sex as described in claim 61, wherein saidmale mammal comprises said bovid, and wherein said concentration ofHoechst 33342 stain is 224 micro-molar.
 63. A method of producing amammal having a predetermined sex as described in claim 61, wherein saidmale mammal comprises said bovid and wherein said concentration ofHoechst 3342 stain is 2240 micro-molar.
 64. A method of producing amammal having a predetermined sex as described in claim 61, furthercomprising the step of adjusting a duration of time said sperm cells areincubated with said concentration of Hoechst 33342 stain between about50 minutes and about 200 minutes.
 65. A method of producing a mammalhaving a predetermined sex as described in claim 62, wherein said stepof adjusting a duration of time said sperm cells are incubated with saidconcentration of Hoechst 33342 stain between about 50 minutes and about200 minutes comprises adjusting said duration of time said sperm cellsare incubated with said concentration of Hoechst 33342 stain to about190 minutes.
 66. A method of producing a mammal having a predeterminedsex as described in claim 63, wherein said step of adjusting a durationof time said sperm cells are incubated with said concentration ofHoechst 33342 stain between about 50 minutes and about 200 minutescomprises adjusting said duration of time said sperm cells are incubatedwith said concentration of Hoechst 33342 stain to about 60 minutes. 67.A method of producing a mammal having a predetermined sex as describedin claims 57, 58, 61, 62, 63, 64, 65, or 66, wherein said step ofstaining DNA within said sperm cells with a concentration of Hoechst33342 greater than 40 micro-molar comprises staining of sufficientuniformity to allow X-chromosome bearing sperm cells to bedifferentiated from Y-chromosome bearing sperm cells based upon themagnitude of fluorescence at a rate of greater than about 85% comprisesa rate selected from the group consisting of 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
 68. A method ofproducing a mammal having a predetermined sex as described in claim 67,wherein said step of staining DNA within said sperm cells withsufficient uniformity to allow X-chromosome bearing sperm cells to bedifferentiated from Y-chromosome bearing sperm cells based upon themagnitude of fluorescence at a rate of greater than about 85% comprisesdifferentiating said magnitude of fluorescence with a flow cytometer.69. A method of staining sperm cells collected from mammals as describedin claim 68, wherein said step of isolating sperm cells separatedaccording to the determination of their sex in a collection elementcomprises isolating Y-chromosome bearing sperm cells into a separatecollection element at a rate of about 1000 per second.
 70. A method ofstaining sperm cells collected from mammals as described in claim 68,wherein said step of isolating sperm cells separated according to thedetermination of their sex in a collection element comprises isolatingX-chromosome bearing sperm cells into a separate collection element at arate of about 1000 per second.
 71. A method of producing a mammal havinga predetermined sex as described in claim 57, further comprising thestep of limiting the number of isolated sperm cells in said artificialinsemination sample to about 10% to about 50% of the number of saidsperm cells relative to a typical unseparated artificial inseminationsample.
 72. A method of producing a mammal having a predetermined sex asdescribed in claim 58, wherein said mammal comprises said bovid andwherein said artificial insemination sample has the number of isolatedsperm cells limited to about one million to three million.
 73. A methodof producing a mammal having a predetermined sex as described in claim58, wherein said mammal is a bovid and wherein said artificialinsemination sample has the number of isolated sperm cells limited tobetween about one-hundred and fifty thousand and about one million. 74.A method of producing a mammal having a predetermined sex as describedin claim 57, wherein said mammal comprises said equid and wherein saidartificial insemination sample has the number of isolated sperm cellslimited to between about forty million and about one hundred million.75. A method of producing a mammal having a predetermined sex asdescribed in claims 71, 72, 73 or 74, further comprising the step ofcreating superovulation in said female mammal to create at least twoeggs comprising the step of using an ovulatory pharmaceutical to causemultiple eggs to be produced, and wherein said ovulatory pharmaceuticalis injected in half day increments between any of days 2 and 18 of theestrus cycle.